Summary Our laboratory recently revealed a critical role for defective viral genomes (DVGs), which are generated during most viral infections, as primary inducers of antiviral immunity in response to many respiratory viral infections. Importantly, detection of DVGs in respiratory secretions from children infected with respiratory syncytial virus (RSV) correlates with evidence of strong antiviral responses, supporting accumulating evidence of a critical role for DVGs in regulating the pathogenesis of RSV. However, study of the biology and clinical impact of DVGs has been slow due, in part, to lack of appropriate technology, and details of the mechanisms that regulate DVG generation and interaction with the host remain unknown. Here we propose to take advantage of important technical advances, including our ability to distinguish DVGs from full-length viral genomes at a single cell level, to significantly improve our understanding of DVG generation and activity during infection with RSV. This pathogen is the major cause of hospitalizations of infants and can cause severe illness and even death in the elderly and the immunosuppressed. Our overarching hypothesis is that DVGs are primary modulators of viral pathogenesis in humans and that their immunostimulatory activity can be harnessed to minimize viral-associated disease. In this proposal, we will follow up on preliminary data challenging the current paradigm that DVG generation initiates randomly during virus replication, and will define viral signatures that regulate the generation of RSV DVGs, thereby advancing strategies to manipulate the DVG content during infection for research and potential therapeutic gain (Aim 1). In addition, we will take advantage of our ability to distinguish DVGs from full-length viral genomes within a cell to understand unique DVG-host interactions that regulate the induction of antiviral immunity during RSV infection (Aim 2). Lastly, we will use unique cohorts of clinical samples readily available to us, to assess the impact of DVGs on the clinical outcome of RSV infection and establish the relationship between DVG levels, disease severity, and host response in humans (Aim 3).